Derangements in apoptosis are common in cancer and confer resistance to cytotoxic therapy. However, the mechanisms that regulate cancer cell death are poorly understood. Dr. Cryns' laboratory has recently identified the heat shock protein alphaB-crystallin as a novel regulator of apoptosis. They have demonstrated that stable expression of alphaB-crystallin in cancer cells confers resistance to chemotherapy-induced apoptosis, at least in part, by a novel mechanism: alphaB-crystallin specifically binds to pro-caspase-3 in vitro and in vivo and inhibits its proteolytic activation by apical caspases. However, caspase-3 is not the only downstream target of alphaB-crystallin: alphaB-crystallin protects MCF-7 breast cancer cells (which lack caspase-3) from chemotherapy-induced apoptosis, although it confers greater protection to MCF-7 cells in which the pro-caspase-3 gene has been stably introduced. Preliminary studies from Dr. Cryns' laboratory also suggest that alphaB-crystallin is phosphorylated in response to chemotherapy and that phosphorylation of alphaB-crystallin may impair its anti-apoptotic function. The goal of the proposed experiments is to examine the hypothesis that alphaB-crystallin expression in cancer cells confers resistance to chemotherapy-induced apoptosis by inhibiting the activation of caspase-3 and other caspase(s). The specific aims are: 1) To determine the molecular mechanisms by which alphaB-crystallin inhibits chemotherapy-induced apoptosis; 2) To delineate the functional domains of alphaB-crystallin that mediate its anti-apoptotic actions; 3) To assess the functional consequences of phosphorylation of alphaB-crystallin; and 4) To determine whether selective inhibition of alphaB-crystallin expression using a deoxyribozyme sensitized cancer cells to chemotherapy-induced caspase activation and apoptosis. These aims will be accomplished using several techniques to quantitatively measure apoptosis (e.g., TUNEL staining and nuclear fragmentation assays) and caspase activation (e.g., pro-caspase proteolytic processing and cleavage of fluorogenic substrates). These studies, then, will provide novel insights into the mechanisms of chemoresistance in cancer and may lead to new therapies for cancer that specifically target alphaB-crystallin.